The primary objective of this investigation is to study the enzymatic mechanisms of DNA metabolism in Escherichia coli and in bacteriophage-infected E. coli. Major emphasis will continue to be on the replication of phage T7 DNA in vivo and in vitro. The nature of the interaction of the gene 5 protein of phage T7 and the E. coli thioredoxin to form an active DNA polymerase will be pursued by in vitro studies using homogeneous proteins. Antibodies to thioredoxin and gene 5 protein and mutationally altered thioredoxin will be employed to determine the role of these proteins in the polymerization reaction and in T7 DNA replication. The gene 4 protein of phage T7 will be purified to homogeneity in order to investigate further the mechanism by which it permits T7 DNA polymerase to copy duplex DNA. Other phage T7 and host proteins involved in T7 DNA replication will be identified, purified, and characterized in order to obtain an extensive synthesis of biologically active duplex DNA in a defined phage system. A separate study involves modification - restriction in the T-even phage system. Studies on the in vitro restriction of DNA containing 5-hydroxymethyldeoxycytosine will be pursued by further purification of the r2,4 protein of E. coli as well as other proteins and factors in this system. The purified enzymes will be used to determine the mechanism of restriction and to identify the restrictive sites in the DNA. Mutants of exonuclease VII will be used to establish the role of this enzyme to DNA metabolism in E. coli and in conjunction with biochemical studies to characterize further its physical and enzymatic properties. DNA polymerases II and III will be used in specific assays to identify other components of bacterial DNA replication. BIBLIOGRAPHIC REFERENCES: Masker, W. E., and Richardson, C.C.: Bacteriophage T7 deoxyribonucleic acid replication in vitro. V. Synthesis of intact chromosomes of bacteriophage T7. J. Mol. Biol., 100, 543-556 (1976). Masker, W.E., and Richardson, C.C.: Bacteriophage T7 deoxyribonucleic acid replication in vitro. VI. Synthesis of biologically active T7 DNA. J. Mol. Biol., 100, 557-567 (1976).